The present invention relates to a new and improved method of reducing scattered light during photometric measurement, especially photometric measurement of haemoglobin, and furthermore, pertains to a new and improved reagent useful for the performance of the aforementioned method.
The invention is in the field of photometry and relates to a method for reducing the scattering of light during photometric measurement, especially during the simultaneous determination of the haemoglobin concentration according to a photometric method and the leukocyte count by an electronic counting operation in a cell suspension which has been prepared by haemolysis and stabilized by a stabilizer.
The counting of red and white blood cells, frequently combined with the determination of the haemoglobin concentration, has been performed for a number of years with partially and fully automated systems. The whole blood which has been rendered noncoagulatable by an anti-coagulant agent, consists of cellular constituents, such as erythrocytes, leukocytes, thrombocytes and plasma containing dissolved constituents therein. For the counting and possible simultaneous size determination of the cellular constituents such must be advantageously prepared for such type analysis. Thus, for instance, the leukocytes and erythrocytes are present in a typical quantitative ratio between one and one thousand. Hence, counting of the leukocytes is appreciably disturbed or even rendered impossible due to the existence of the erythrocytes which prevail in a predominant number. Techniques are utilized which bring about an extensive elimination of the erythrocytes, without changing the quantity of leukocytes. One heretofore known procedure is haemolysation which destroys the form of the erythrocytes, that is to say, splits them into small pieces, without altering the leukocyte count. During the counting, for instance, according to electrical resistance methods, it is possible to detect the erythrocyte fragments as artifacts and to distinguish them from the count of the leukocytes. The haemoglobin which is released during the destruction of the erythrocytes can be ancillarily determined photometrically, through the addition of further reagents.
The simultaneous determination of the leukocytes and the haemoglobin concentration of samples which have been prepared in this manner is limited in time, normally such samples remain stable for about 20 minutes, and therefore, within this period of time it is necessary to perform the analysis.
A typical characteristic of the haemolytic methods is that unspecific turbidity arises owing to the large quantity of the fragments of the erythrocytes which are present in the solution, but also because of the leukocytes which remain intact and which are to be counted, and such has a disturbing effect during the photometric measurement of the haemoglobin. It is known from nephelometry that the light intensity, transmitted by the scattering solution, is proportional to the number of colloid particles and to the square of their volume (RAYLEIGH). The cell dissolution into fragments of different size and number, caused by haemolysis, thus produces an uncontrollable transmission loss of the irradiated suspension. The proportion of light in the blue spectral region, which proportion has been reduced by the dispersion, is not a major factor during haemoglobin measurement. If the formed cell fragments and also the morphologically altered leukocytes further change their volume following haemolysis, then this transmission loss additionally is dependent upon time. The measured extinction thus is associated with a non-systematic and time-dependent error in the form of an additional pseudo extinction and cannot be satisfactorily compensated by callibration operations. This fault can amount to as much as 3% to 7% of the haemoglobin value.